EP3365366B1 - Protéines de liaison trispécifiques et/ou trivalentes pour la prévention ou le traitement d'une infection par le vih - Google Patents

Protéines de liaison trispécifiques et/ou trivalentes pour la prévention ou le traitement d'une infection par le vih Download PDF

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EP3365366B1
EP3365366B1 EP16794472.7A EP16794472A EP3365366B1 EP 3365366 B1 EP3365366 B1 EP 3365366B1 EP 16794472 A EP16794472 A EP 16794472A EP 3365366 B1 EP3365366 B1 EP 3365366B1
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seq
sequence
cdr
amino acid
artificial sequence
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EP3365366A1 (fr
Inventor
Zhi-Yong Yang
Gary J. Nabel
Ling Xu
Ronnie WEI
Huawei Qiu
Jochen Beninga
Jochen Kruip
Ercole Rao
Wulf Dirk LEUSCHNER
Christian Beil
Christian Lange
Mark Connors
John R. Mascola
Richard A. Koup
Jinghe HUANG
Nicole A. DORIA-ROSE
Tongqing Zhou
Peter D. Kwong
Young Do KWON
Amarendra PEGU
Mangaiarkarasi ASOKAN
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Sanofi SA
US Department of Health and Human Services
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Sanofi SA
US Department of Health and Human Services
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Priority to PL16794472T priority Critical patent/PL3365366T3/pl
Priority to RS20211266A priority patent/RS62437B1/sr
Priority to SI201631358T priority patent/SI3365366T1/sl
Priority to HRP20211528TT priority patent/HRP20211528T1/hr
Priority to EP20197556.2A priority patent/EP3819310A1/fr
Publication of EP3365366A1 publication Critical patent/EP3365366A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6881—Cluster-antibody conjugates, i.e. the modifying agent consists of a plurality of antibodies covalently linked to each other or of different antigen-binding fragments covalently linked to each other
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the disclosure relates to trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein a first pair of polypeptides forming the binding protein possess dual variable domains having a cross-over orientation and wherein a second pair of polypeptides forming the binding protein possess a single variable domain.
  • the disclosure also relates to methods for making trispecific and/or trivalent binding proteins and uses of such binding proteins for treating and/or preventing HIV/AIDS.
  • HIV/AIDS neutralizing antibodies
  • virological events in the early weeks following HIV-1 transmission set the stage for lifelong chronic infection that remains incurable with currently available combination antiretroviral therapy (cART). This is due, at least in part, to the early establishment of viral reservoirs, including latently infected cells, which persist despite cART, leading to recrudescent infection when treatment is interrupted.
  • cART combination antiretroviral therapy
  • Newly discovered anti-HIV-1 neutralizing antibodies with improved breadth and potency may provide more options for HIV/AIDS treatment and prevention; however, breakthrough infection remains a major issue in the field.
  • WO 2012/158948 discloses an isolated HIV antibody. However, WO 2012/158948 does not provide or mention tri-specific antibodies.
  • the disclosure provides a binding protein comprising four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein a first polypeptide chain comprises a structure represented by the formula: V L2 - L 1 -V L1 - L 2 -C L [I]; a second polypeptide chain comprises a structure represented by the formula: V H1 - L 3 -V H2 - L 4 -CH 1 [II]; a third polypeptide chain comprises a structure represented by the formula: V H3- C H1 [III]; and a fourth polypeptide chain comprises a structure represented by the formula: V L3 -C L [IV]; wherein
  • the binding protein of the present invention comprises
  • the binding protein of the present invention is characterized in that
  • the C H3 domains of the second and the third polypeptide chains both comprise amino acid substitutions at positions corresponding to positions 428 and 434 of human IgG1 according to EU Index, wherein the amino acid substitutions are M428L and N434S.
  • L 1 , L 2 , L 3 , or L 4 is independently 0 amino acids in length; or (b) L 1 , L 2 , L 3 , or L 4 are each independently at least one amino acid in length.
  • L 1 , L 2 , L 3 , and/or L 4 comprise the sequence Asp-Lys-Thr-His-Thr (SEQ ID NO: 525).
  • the present disclosure provides a binding protein comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain and a fourth polypeptide chain wherein:
  • the present disclosure provides an expression vector comprising the nucleic acid molecule of the present invention.
  • the present disclosure provided vector system comprising one or more vectors encoding a first, second, third, and fourth polypeptide chain of a binding protein of the present invention, wherein optionally:
  • the present disclosure provides isolated host cell comprising the nucleic acid molecule of the present invention, the expression vector the present invention, or the vector system of the present invention, wherein optionally the host cell is a mammalian cell or an insect cell.
  • the present disclosure provides a method of producing a binding protein, the method comprising:
  • the present disclosure provided a binding protein of the present invention for use in a method of preventing and/or treating HIV infection in a patient, comprising administering to the patient a therapeutically effective amount of the binding protein, wherein the binding protein is optionally co-administered with standard anti-retroviral therapy, and wherein the patient is optionally a human.
  • the present disclosure provides trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind to one or more human immunodeficiency virus (HIV) target proteins and/or one or more T-cell receptor target proteins, wherein a first pair of polypeptides forming the binding protein possess dual variable domains having a cross-over orientation and wherein a second pair of polypeptides forming the binding protein possess a single variable domain.
  • HIV human immunodeficiency virus
  • polynucleotide refers to single-stranded or double-stranded nucleic acid polymers of at least 10 nucleotides in length.
  • the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
  • Such modifications include base modifications such as bromuridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.
  • base modifications such as bromuridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.
  • polynucleotide specifically includes single-stranded and double-stranded forms of DNA.
  • isolated polynucleotide is a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which: (1) is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.
  • isolated polypeptide is one that: (1) is free of at least some other polypeptides with which it would normally be found, (2) is essentially free of other polypeptides from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a polypeptide with which the "isolated polypeptide" is associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature.
  • Such an isolated polypeptide can be encoded by genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof.
  • the isolated polypeptide is substantially free from polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).
  • Naturally occurring antibodies typically comprise a tetramer.
  • Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length "light” chain (typically having a molecular weight of about 25 kDa) and one full-length "heavy” chain (typically having a molecular weight of about 50-70 kDa).
  • the terms "heavy chain” and “light chain” as used herein refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen.
  • the amino-terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids that typically is responsible for antigen recognition.
  • the carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function.
  • a full-length heavy chain immunoglobulin polypeptide includes a variable domain (V H ) and three constant domains (C H1 , C H2 , and C H3 ), wherein the V H domain is at the amino-terminus of the polypeptide and the C H3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide includes a variable domain (V L ) and a constant domain (C L ), wherein the V L domain is at the amino-terminus of the polypeptide and the C L domain is at the carboxyl-terminus.
  • Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4.
  • IgM has subclasses including, but not limited to, IgM1 and IgM2.
  • IgA is similarly subdivided into subclasses including, but not limited to, IgA1 and IgA2.
  • variable and constant domains typically are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids. See, e.g., FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd ed., 1989 ).
  • the variable regions of each light/heavy chain pair typically form an antigen binding site.
  • the variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs.
  • the CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope. From the amino-terminus to the carboxyl-terminus, both light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • CDR set refers to a group of three CDRs that occur in a single variable region capable of binding the antigen.
  • the exact boundaries of these CDRs have been defined differently according to different systems.
  • the system described by Kabat Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (National Institutes of Health, Bethesda, Md. (1987 ) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs.
  • These CDRs may be referred to as Kabat CDRs. Chothia and coworkers ( Chothia and Lesk, 1987, J. Mol. Biol.
  • CDR boundary definitions may not strictly follow one of the herein systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding.
  • the methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia defined CDRs. Identification of predicted CDRs using the amino acid sequence is well known in the field, such as in Martin, A.C. "Protein sequence and structure analysis of antibody variable domains," In Antibody Engineering, Vol. 2.
  • the amino acid sequence of the heavy and/or light chain variable domain may be also inspected to identify the sequences of the CDRs by other conventional methods, e.g ., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
  • the numbered sequences may be aligned by eye, or by employing an alignment program such as one of the CLUSTAL suite of programs, as described in Thompson, 1994, Nucleic Acids Res. 22: 4673-80 .
  • Molecular models are conventionally used to correctly delineate framework and CDR regions and thus correct the sequence-based assignments.
  • Fc refers to a molecule comprising the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region.
  • the original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins, although IgG1 and IgG2 are preferred.
  • Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent ( i.e., disulfide bonds) and non-covalent association.
  • the number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2).
  • class e.g., IgG, IgA, and IgE
  • subclass e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2
  • Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG.
  • Fc as used herein is generic to the monomeric, dimeric, and multimeric forms.
  • a F(ab) fragment typically includes one light chain and the V H and C H1 domains of one heavy chain, wherein the V H -C H1 heavy chain portion of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide.
  • a F(ab) fragment can also include one light chain containing two variable domains separated by an amino acid linker and one heavy chain containing two variable domains separated by an amino acid linker and a C H1 domain.
  • a F(ab') fragment typically includes one light chain and a portion of one heavy chain that contains more of the constant region (between the C H1 and C H2 domains), such that an interchain disulfide bond can be formed between two heavy chains to form a F(ab')2 molecule.
  • binding protein refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds to at least one target antigen, and which comprises four polypeptide chains that form at least three antigen binding sites, wherein a first polypeptide chain has a structure represented by the formula: V L2 - L 1 -V L1 - L 2 -C L [I] and a second polypeptide chain has a structure represented by the formula: V H1 - L 3 -V H2 - L 4 -CH 1 [II] and a third polypeptide chain has a structure represented by the formula: V H3 -C H1 [III] and a fourth polypeptide chain has a structure represented by the formula: V L3 -C L [IV] wherein:
  • a "recombinant" molecule is one that has been prepared, expressed, created, or isolated by recombinant means.
  • binding proteins having biological and immunological specificity to between one and three target antigens.
  • nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins.
  • expression vectors comprising nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins.
  • host cells that express such binding proteins (i.e., comprising nucleic acid molecules or vectors encoding polypeptide chains that form such binding proteins).
  • variable domains refers to the interchangeability of variable domains within the binding protein format and with retention of folding and ultimate binding affinity.
  • “Full swapability” refers to the ability to swap the order of both V H1 and V H2 domains, and therefore the order of V L1 and V L2 domains, in the polypeptide chain of formula I or the polypeptide chain of formula II ( i.e., to reverse the order) while maintaining full functionality of the binding protein as evidenced by the retention of binding affinity.
  • V H and V L refer only to the domain's location on a particular protein chain in the final format.
  • V H1 and V H2 could be derived from V L1 and V L2 domains in parent antibodies and placed into the V H1 and V H2 positions in the binding protein.
  • V L1 and V L2 could be derived from V H1 and V H2 domains in parent antibodies and placed in the V H1 and V H2 positions in the binding protein.
  • the V H and V L designations refer to the present location and not the original location in a parent antibody. V H and V L domains are therefore "swappable.”
  • antigen or “target antigen” or “antigen target” as used herein refers to a molecule or a portion of a molecule that is capable of being bound by a binding protein, and additionally is capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen.
  • a target antigen may have one or more epitopes. With respect to each target antigen recognized by a binding protein, the binding protein is capable of competing with an intact antibody that recognizes the target antigen.
  • HIV Human Immunodeficiency Virus
  • HIV infection generally encompasses infection of a host, particularly a human host, by the human immunodeficiency virus (HIV) family of retroviruses including, but not limited to, HIV I, HIV II, HIV III (also known as HTLV-II, LAV-1, LAV-2). HIV can be used herein to refer to any strains, forms, subtypes, clades and variations in the HIV family.
  • HIV infection will encompass the treatment of a person who is a carrier of any of the HIV family of retroviruses or a person who is diagnosed with active AIDS, as well as the treatment or prophylaxis of the AIDS-related conditions in such persons.
  • AIDS as used herein means Acquired Immunodeficiency Syndrome. AIDS is caused by HIV.
  • CD4bs or “CD4 binding site” refer to the binding site for CD4 (cluster of differentiation 4), which is a glycoprotein found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells.
  • CD3 is cluster of differentiation factor 3 polypeptide and is a T-cell surface protein that is typically part of the T cell receptor (TCR) complex.
  • CD28 is cluster of differentiation 28 polypeptide and is a T-cell surface protein that provides co-stimulatory signals for T-cell activation and survival.
  • glycoprotein 160 or "gp160 protein” refers to the envelope glycoprotein complex of HIV and which is a homotrimer that is cleaved into gp120 and gp41 subunits.
  • MPER refers to the membrane-proximal external region of glycoprotein 41 (gp41), which is a subunit of the envelope protein complex of retroviruses, including HIV.
  • glycocan refers to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan.
  • glycan refers to the HIV-1 envelope glycoprotein gp120.
  • T-cell engager refers to binding proteins directed to a host's immune system, more specifically the T cells' cytotoxic activity as well as directed to a HIV target protein.
  • trimer apex refers to apex of HIV-1 envelope glycoprotein gp120.
  • the term "monospecific binding protein” refers to a binding protein that specifically binds to one antigen target.
  • binding protein refers to a binding protein that has one antigen binding site.
  • binding protein refers to a binding protein that specifically binds to two different antigen targets.
  • bivalent binding protein refers to a binding protein that has two binding sites.
  • trispecific binding protein refers to a binding protein that specifically binds to three different antigen targets.
  • trivalent binding protein refers to a binding protein that has three binding sites. In particular embodiments the trivalent binding protein can bind to one antigen target. In other embodiments, the trivalent binding protein can bind to two antigen targets. In other embodiments, the trivalent binding protein can bind to three antigen targets.
  • an "isolated" binding protein is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the binding protein, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the binding protein will be purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated binding proteins include the binding protein in situ within recombinant cells since at least one component of the binding protein's natural environment will not be present.
  • substantially pure or substantially purified refer to a compound or species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition).
  • a substantially purified fraction is a composition wherein the species comprises at least about 50% (on a molar basis) of all macromolecular species present.
  • a substantially pure composition will comprise more than about 80%, 85%, 90%, 95%, or 99% of all macromolar species present in the composition.
  • the species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
  • a “neutralizing" binding protein as used herein refers to a molecule that is able to block or substantially reduce an effector function of a target antigen to which it binds.
  • substantially reduce means at least about 60%, preferably at least about 70%, more preferably at least about 75%, even more preferably at least about 80%, still more preferably at least about 85%, most preferably at least about 90% reduction of an effector function of the target antigen.
  • epitope includes any determinant, preferably a polypeptide determinant, capable of specifically binding to an immunoglobulin or T-cell receptor.
  • epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics and/or specific charge characteristics.
  • An epitope is a region of an antigen that is bound by an antibody or binding protein.
  • a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
  • a binding protein is said to specifically bind an antigen when the equilibrium dissociation constant is ⁇ 10 -8 M, more preferably when the equilibrium dissociation constant is ⁇ 10 -9 M, and most preferably when the dissociation constant is ⁇ 10 -10 M.
  • the dissociation constant (K D ) of a binding protein can be determined, for example, by surface plasmon resonance.
  • surface plasmon resonance analysis measures real-time binding interactions between ligand (a target antigen on a biosensor matrix) and analyte (a binding protein in solution) by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor; Piscataway, NJ).
  • SPR surface plasmon resonance
  • Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen).
  • K D refers to the dissociation constant of the interaction between a particular binding protein and a target antigen.
  • the term "specifically binds" as used herein refers to the ability of a binding protein or an antigen-binding fragment thereof to bind to an antigen containing an epitope with an Kd of at least about 1 x 10 -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, 1 x 10 -12 M, or more, and/or to bind to an epitope with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen.
  • linker refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient mobility for the domains of the light and heavy chains to fold into cross over dual variable region immunoglobulins.
  • a linker is inserted at the transition between variable domains or between variable and constant domains, respectively, at the sequence level.
  • the transition between domains can be identified because the approximate size of the immunoglobulin domains are well understood.
  • the precise location of a domain transition can be determined by locating peptide stretches that do not form secondary structural elements such as beta-sheets or alpha-helices as demonstrated by experimental data or as can be assumed by techniques of modeling or secondary structure prediction.
  • the linkers described herein are referred to as L 1 , which is located on the light chain between the C-terminus of the V L2 and the N-terminus of the V L1 domain; and L 2 , which is located on the light chain between the C-terminus of the V L1 and the N-terminus of the C L domain.
  • the heavy chain linkers are known as L 3 , which is located between the C-terminus of the V H1 and the N-terminus of the V H2 domain; and L 4 , which is located between the C-terminus of the V H2 and the N-terminus of the C H1 domain.
  • vector refers to any molecule (e.g., nucleic acid, plasmid, or virus) that is used to transfer coding information to a host cell.
  • the term “vector” includes a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double-stranded DNA molecule into which additional DNA segments may be inserted.
  • viral vector Another type of vector, wherein additional DNA segments may be inserted into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. , bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g. , non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "recombinant expression vectors” (or simply, “expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably herein, as a plasmid is the most commonly used form of vector.
  • the disclosure is intended to include other forms of expression vectors, such as viral vectors (e.g. , replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.
  • recombinant host cell refers to a cell into which a recombinant expression vector has been introduced.
  • a recombinant host cell or host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but such cells are still included within the scope of the term "host cell” as used herein.
  • host cell expression systems can be used to express the binding proteins, including bacterial, yeast, baculoviral, and mammalian expression systems (as well as phage display expression systems).
  • a suitable bacterial expression vector is pUC19.
  • a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chains of the binding protein such that the polypeptide chains are expressed in the host cell and, preferably, secreted into the medium in which the host cells are cultured, from which medium the binding protein can be recovered.
  • transformation refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain a new DNA.
  • a cell is transformed where it is genetically modified from its native state.
  • the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, or may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid.
  • a cell is considered to have been stably transformed when the DNA is replicated with the division of the cell.
  • transfection refers to the uptake of foreign or exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are well known in the art. Such techniques can be used to introduce one or more exogenous DNA molecules into suitable host cells.
  • non-naturally occurring refers to the fact that the object can be found in nature and has not been manipulated by man.
  • a polynucleotide or polypeptide that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man is naturally-occurring.
  • non-naturally occurring refers to an object that is not found in nature or that has been structurally modified or synthesized by man.
  • the twenty conventional amino acids and their abbreviations follow conventional usage.
  • Stereoisomers e.g. , D-amino acids
  • unnatural amino acids and analogs such as ⁇ -, ⁇ -disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for the polypeptide chains of the binding proteins.
  • Examples of unconventional amino acids include: 4-hydroxyproline, ⁇ -carboxyglutamate, ⁇ -N,N,N-trimethyllysine, ⁇ -N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ⁇ -N-methylarginine, and other similar amino acids and imino acids (e.g. , 4-hydroxyproline).
  • the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention.
  • Naturally occurring residues may be divided into classes based on common side chain properties:
  • Conservative amino acid substitutions may involve exchange of a member of one of these classes with another member of the same class.
  • Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.
  • a skilled artisan will be able to determine suitable variants of the polypeptide chains of the binding proteins using well-known techniques. For example, one skilled in the art may identify suitable areas of a polypeptide chain that may be changed without destroying activity by targeting regions not believed to be important for activity. Alternatively, one skilled in the art can identify residues and portions of the molecules that are conserved among similar polypeptides. In addition, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
  • patient includes human and animal subjects.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures.
  • Those in need of treatment include those having the disorder as well as those prone to have a disorder or those in which the disorder is to be prevented.
  • binding proteins can be used to treat humans infected with HIV, or humans susceptible to HIV infection, or ameliorate HIV infection in a human subject infected with HIV.
  • the binding proteins can also be used to prevent HIV in a human patient.
  • treating humans infected with HIV include those subjects who are at any one of the several stages of HIV infection progression, which, for example, include acute primary infection syndrome (which can be asymptomatic or associated with an influenza-like illness with fevers, malaise, diarrhea and neurologic symptoms such as headache), asymptomatic infection (which is the long latent period with a gradual decline in the number of circulating CD4 + T cells), and AIDS (which is defined by more serious AIDS-defining illnesses and/or a decline in the circulating CD4 cell count to below a level that is compatible with effective immune function).
  • acute primary infection syndrome which can be asymptomatic or associated with an influenza-like illness with fevers, malaise, diarrhea and neurologic symptoms such as headache
  • asymptomatic infection which is the long latent period with a gradual decline in the number of circulating CD4 + T cells
  • AIDS which is defined by more serious AIDS-defining illnesses and/or a decline in the circulating CD4 cell count to below a level that is compatible with effective immune function).
  • treating or preventing HIV infection will also encompass treating suspected infection by HIV after suspected past exposure to HIV by e.g., contact with HIV-contaminated blood, blood transfusion, exchange of body fluids, "unsafe" sex with an infected person, accidental needle stick, receiving a tattoo or acupuncture with contaminated instruments, or transmission of the virus from a mother to a baby during pregnancy, delivery or shortly thereafter.
  • composition or “therapeutic composition” as used herein refer to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.
  • pharmaceutically acceptable carrier or “physiologically acceptable carrier” as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of a binding protein.
  • a therapeutically effective amount when used in reference to a pharmaceutical composition comprising one or more binding proteins refer to an amount or dosage sufficient to produce a desired therapeutic result. More specifically, a therapeutically effective amount is an amount of a binding protein sufficient to inhibit, for some period of time, one or more of the clinically defined pathological processes associated with the condition being treated. The effective amount may vary depending on the specific binding protein that is being used, and also depends on a variety of factors and conditions related to the patient being treated and the severity of the disorder. For example, if the binding protein is to be administered in vivo, factors such as the age, weight, and health of the patient as well as dose response curves and toxicity data obtained in preclinical animal work would be among those factors considered. The determination of an effective amount or therapeutically effective amount of a given pharmaceutical composition is well within the ability of those skilled in the art.
  • One embodiment of the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein.
  • the binding protein of the disclosure is a trispecific and/or trivalent binding protein comprising four polypeptide chains that form three antigen binding sites that specifically bind one or more (e.g., three different) HIV target proteins, wherein a first polypeptide chain comprises a structure represented by the formula: V L2 - L 1 -V L1 - L 2 -C L [I] and a second polypeptide chain comprises a structure represented by the formula: V H1 - L 3 -V H2 - L 4 -CH 1 [II] and a third polypeptide chain comprises a structure represented by the formula: V H3 -C H1 [III] and a fourth polypeptide chain comprises a structure represented by the formula: V L3 -C L [IV] wherein:
  • the binding protein of the present invention comprises
  • the binding protein of the present invention is characterized in that
  • the C H3 domains of the second and the third polypeptide chains both comprise amino acid substitutions at positions corresponding to positions 428 and 434 of human IgG1 according to EU Index, wherein the amino acid substitutions are M428L and N434S.
  • L 1 , L 2 , L 3 , or L 4 is independently 0 amino acids in length; or (b) L 1 , L 2 , L 3 , or L 4 are each independently at least one amino acid in length.
  • L 1 , L 2 , L 3 , and/or L 4 comprise the sequence Asp-Lys-Thr-His-Thr (SEQ ID NO: 525).
  • the present disclosure provides a binding protein comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain and a fourth polypeptide chain wherein:
  • the first polypeptide chain and the second polypeptide chain have a cross-over orientation that forms two distinct antigen binding sites.
  • the V H1 and V L1 form a binding pair and form the first antigen binding site.
  • the V H2 and V L2 form a binding pair and form the second antigen binding site.
  • the third polypeptide and the fourth polypeptide form a third antigen binding site.
  • the V H3 and V L3 form a binding pair and form the third antigen binding site.
  • the order of the V H1 and V H2 domains, and therefore the order of V L1 and V L2 domains, in the polypeptide chain of formula I or the polypeptide chain of formula II are swapped.
  • the binding proteins specifically bind to one or more HIV target proteins.
  • the binding proteins are trispecific and specifically bind to MPER of the HIV-1 gp41 protein, a CD4 binding site of the HIV-1 gp120 protein, a glycan in the V3 loop of the HIV-1 gp120 protein, a trimer apex of the HIV-1 gp120 protein or gp160.
  • the binding proteins specifically bind to one or more HIV target proteins and one or more target proteins on a T-cell including T cell receptor complex. These T-cell engager binding proteins are capable of recruiting T cells transiently to target cells and, at the same time, activating the cytolytic activity of the T cells.
  • the T-cell engager trispecific antibodies can be used to activate HIV-1 reservoirs and redirect/activate T cells to lyse latently infected HIV-1 + T cells.
  • target proteins on T cells include but are not limited to CD3 and CD28, among others.
  • the trispecific binding proteins may be generated by combining the antigen binding domains of two or more monospecific antibodies (parent antibodies) into one antibody. See International Publication Nos.
  • binding proteins of the disclosure may be prepared using domains or sequences obtained or derived from any human or non-human antibody, including, for example, human, murine, or humanized antibodies.
  • the trivalent binding protein is capable of binding three different antigen targets.
  • the binding protein is trispecific and one light chain-heavy chain pair is capable of binding two different antigen targets or epitopes and one light chain-heavy chain pair is capable of binding one antigen target or epitope.
  • the binding protein is capable of binding three different HIV antigen targets that are located on the HIV envelope glycoprotein structure composed of gp120 and gp41 subunits.
  • the binding protein is capable of inhibiting the function of one or more of the antigen targets.
  • a binding protein of the present disclosure binds one or more HIV target proteins.
  • the binding protein is capable of specifically binding three different epitopes on a single HIV target protein.
  • the binding protein is capable of binding two different epitopes on a first HIV target protein, and one epitope on a second HIV target protein.
  • the first and second HIV target proteins are different.
  • the binding protein is capable of specifically binding three different HIV target protein.
  • the one or more HIV target proteins are one or more of glycoprotein 120, glycoprotein 41, and glycoprotein 160.
  • a binding protein of the present disclosure binds one or more HIV target proteins and one or more T cell target proteins.
  • the binding protein is capable of specifically binding one HIV target protein and two different epitopes on a single T cell target protein.
  • the binding protein is capable of specifically binding one HIV target protein and two different T cell target proteins (e.g., CD28 and CD3).
  • the binding protein is capable of specifically binding one T cell target protein and two different epitopes on a single HIV target protein.
  • the binding protein is capable of specifically binding one T cell target protein and two different HIV target proteins.
  • the first and second polypeptide chains of the binding protein form two antigen binding sites that specifically target two T cell target proteins, and the third and fourth polypeptide chains of the binding protein form an antigen binding site that specifically binds an HIV target protein.
  • the one or more HIV target proteins are one or more of glycoprotein 120, glycoprotein 41, and glycoprotein 160.
  • the one or more T cell target proteins are one or more of CD3 and CD28.
  • the linkers can also be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids long.
  • L 1 , L 2 , L 3 , and L4 in one binding protein may all have the same amino acid sequence or may all have different amino acid sequences.
  • linkers include a single glycine (Gly) residue; a diglycine peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with four glycine residues (Gly-Gly-Gly-Gly; SEQ ID NO: 285); a peptide with five glycine residues (Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 286); a peptide with six glycine residues (Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 287); a peptide with seven glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 288); a peptide with eight glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 289).
  • amino acid residues may be used such as the peptide Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 290), the peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 291) and the peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 292).
  • linkers include a single Ser, and Val residue; the dipeptide Arg-Thr, Gln-Pro, Ser-Ser, Thr-Lys, and Ser-Leu; Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 293), Thr-Val-Ala-Ala-Pro (SEQ ID NO: 294), Gln-Pro-Lys-Ala-Ala (SEQ ID NO: 295), Gln-Arg-Ile-Glu-Gly (SEQ ID NO: 296); Ala-Ser-Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 297), Arg-Thr-Val-Ala-Ala-Pro-Ser (SEQ ID NO: 298), Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 299), and His-Ile-Asp-Ser-Pro-Asn-Lys (SEQ ID NO: 300).
  • Linkers comprising randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline have been shown to be suitable in the binding proteins.
  • the identity and sequence of amino acid residues in the linker may vary depending on the type of secondary structural element necessary to achieve in the linker. For example, glycine, serine, and alanine are best for linkers having maximum flexibility. Some combination of glycine, proline, threonine, and serine are useful if a more rigid and extended linker is necessary. Any amino acid residue may be considered as a linker in combination with other amino acid residues to construct larger peptide linkers as necessary depending on the desired properties.
  • the length of L 1 is at least twice the length of L 3 . In some embodiments, the length of L 2 is at least twice the length of L 4 . In some embodiments, the length of L 1 is at least twice the length of L 3 , and the length of L 2 is at least twice the length of L 4 . In some embodiments, L 1 is 3 to 12 amino acid residues in length, L 2 is 3 to 14 amino acid residues in length, L 3 is 1 to 8 amino acid residues in length, and L 4 is 1 to 3 amino acid residues in length.
  • L 1 is 5 to 10 amino acid residues in length
  • L 2 is 5 to 8 amino acid residues in length
  • L 3 is 1 to 5 amino acid residues in length
  • L 4 is 1 to 2 amino acid residues in length.
  • L 1 is 7 amino acid residues in length
  • L 2 is 5 amino acid residues in length
  • L 3 is 1 amino acid residue in length
  • L 4 is 2 amino acid residues in length.
  • L 1 , L 2 , L 3 , and/or L 4 comprise the sequence Asp-Lys-Thr-His-Thr (SEQ ID NO: 525).
  • L 1 comprises the sequence Asp-Lys-Thr-His-Thr (SEQ ID NO: 525).
  • L 3 comprises the sequence Asp-Lys-Thr-His-Thr (SEQ ID NO: 525).
  • L 1 , L 2 , L 3 , and/or L 4 comprise the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 299).
  • L 1 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 299).
  • L 1 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 299)
  • L 2 comprises the sequence Thr-Lys-Gly-Pro-Ser-Arg (SEQ ID NO: 526)
  • L 3 comprises the sequence Ser
  • L 4 comprises the sequence Arg-Thr.
  • L 3 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 299).
  • L 1 comprises the sequence Ser
  • L 2 comprises the sequence Arg-Thr
  • L 3 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 299)
  • L 4 comprises the sequence Thr-Lys-Gly-Pro-Ser-Arg (SEQ ID NO: 526).
  • a binding protein of the present disclosure comprises a second polypeptide chain further comprising an Fc region linked to C H1 , the Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains.
  • a binding protein of the present disclosure comprises a third polypeptide chain further comprising an Fc region linked to C H1 , the Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains.
  • a binding protein of the present disclosure comprises a second polypeptide chain further comprising an Fc region linked to C H1 , the Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising an Fc region linked to C H1 , the Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains.
  • the C H3 domains can be altered by the "knob-into-holes" technology which is described in detail with several examples in, for example, International Publication No. WO 96/027011 , Ridgway et al., 1996, Protein Eng. 9: 617-21 ; and Merchant et al., 1998, Nat. Biotechnol. 16: 677-81 .
  • the interaction surfaces of the two C H3 domains are altered to increase the heterodimerisation of both heavy chains containing these two C H3 domains.
  • Each of the two C H3 domains can be the "knob," while the other is the “hole.”
  • the introduction of a disulfide bridge further stabilizes the heterodimers (Merchant et al., 1998; Atwell et al., 1997, J. Mol. Biol. 270: 26-35 ) and increases the yield.
  • the knob is on the second pair of polypeptides with a single variable domain.
  • the knob is on the first pair of polypeptides having the cross-over orientation.
  • the C H3 domains do not include a knob in hole.
  • a binding protein of the present disclosure comprises a "knob” mutation on the second polypeptide chain and a "hole” mutation on the third polypeptide chain. In some embodiments, a binding protein of the present disclosure comprises a "knob” mutation on the third polypeptide chain and a "hole” mutation on the second polypeptide chain. In some embodiments, the "knob” mutation comprises substitutions at positions corresponding to positions 354 and 366 of human IgG1 according to EU Index. In some embodiments, the amino acid substitutions are S354C and T366W. In some embodiments, the "hole” mutation comprises substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 according to EU Index.
  • the amino acid substitutions are Y349C, T366S, L368A, and Y407V.
  • the second polypeptide chain further comprises a first Fc region linked to C H1 , the first Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to C H1 , the second Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 according to EU Index, wherein the amino acid substitutions are Y349C,
  • the second polypeptide chain further comprises a first Fc region linked to C H1 , the first Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to C H1 , the second Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 according to EU Index, wherein the amino acid substitutions are S354C and T366W.
  • a binding protein of the present disclosure comprises one or more mutations to improve serum half-life ( See e.g., Hinton, P.R. et al. (2006) J. Immunol. 176(1):346-56 ).
  • the mutation comprises substitutions at positions corresponding to positions 428 and 434 of human IgG1 according to EU Index, wherein the amino acid substitutions are M428L and N434S.
  • the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to C H1 , the first Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to C H1 , the second Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains, wherein the first and second Fc regions comprise amino acid substitutions at positions corresponding to positions 428 and 434 of human IgG1 according to EU Index, wherein the amino acid substitutions are M428L and N434S.
  • a binding protein of the present disclosure comprises knob and hole mutations and one or more mutations to improve serum half-life.
  • C H1 , C H2 , C H3 and C L of the trispecific binding proteins described herein may comprise any of C H1 , C H2 , C H3 and C L sequences of binding proteins 1-53.
  • Standard recombinant DNA methodologies are used to construct the polynucleotides that encode the polypeptides which form the binding proteins, incorporate these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. See e.g., Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 3rd ed .). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein.
  • isolated nucleic acid molecules comprising a nucleotide sequence encoding any of the binding proteins described herein.
  • the isolated nucleic acid is operably linked to a heterologous promoter to direct transcription of the binding protein-coding nucleic acid sequence.
  • a promoter may refer to nucleic acid control sequences which direct transcription of a nucleic acid.
  • a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence of a binding protein if the promoter affects the transcription or expression of the coding sequence.
  • promoters may include, but are not limited to, promoters obtained from the genomes of viruses (such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40 (SV40), and the like), from heterologous eukaryotic promoters (such as the actin promoter, an immunoglobulin promoter, from heat-shock promoters, and the like), the CAG-promoter ( Niwa et al., Gene 108(2):193-9, 1991 ), the phosphoglycerate kinase (PGK)-promoter, a tetracycline-inducible promoter ( Masui et al., Nucleic Acids Res.
  • viruses such as polyoma virus, fowlpox virus, adeno
  • polynucleotides encoding binding proteins of the present disclosure may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein or other suitable promoter that will be readily recognized by one skilled in the art.
  • the isolated nucleic acid is incorporated into a vector.
  • the vector is an expression vector.
  • Expression vectors may include one or more regulatory sequences operatively linked to the polynucleotide to be expressed.
  • regulatory sequence includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals).
  • Suitable enhancers may include, but are not limited to, enhancer sequences from mammalian genes (such as globin, elastase, albumin, ⁇ -fetoprotein, insulin and the like), and enhancer sequences from a eukaryotic cell virus (such as SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, and the like).
  • mammalian genes such as globin, elastase, albumin, ⁇ -fetoprotein, insulin and the like
  • enhancer sequences from a eukaryotic cell virus such as SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, and the like).
  • suitable vectors may include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenoviral, vaccinia viral, Sindbis-viral, measles, herpes viral, lentiviral, retroviral, adeno-associated viral vectors, etc.).
  • Expression vectors can be used to transfect host cells, such as, for example, bacterial cells, yeast cells, insect cells, and mammalian cells.
  • Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art, and can be used to transfect any cell of interest.
  • the vector system comprises one or more vectors encoding a first, second, third, and fourth polypeptide chain of any of the binding proteins described herein.
  • the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein.
  • the vector system comprises a first vector encoding the first and second polypeptide chains of the binding protein, and a second vector encoding the third and fourth polypeptide chains of the binding protein.
  • the vector system comprises a first vector encoding the first and third polypeptide chains of the binding protein, and a second vector encoding the second and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and fourth polypeptide chains of the binding protein, and a second vector encoding the second and third polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first, second, third, and fourth polypeptide chains of the binding protein.
  • the one or more vectors of the vector system may be any of the vectors described herein. In some embodiments, the one or more vectors are expression vectors.
  • a host cell e.g., an isolated host cell
  • an isolated host cell of the present disclosure is cultured in vitro.
  • the host cell is a bacterial cell (e.g., an E. coli cell).
  • the host cell is a yeast cell (e.g., an S. cerevisiae cell).
  • the host cell is an insect cell.
  • insect host cells may include, for example, Drosophila cells (e.g., S2 cells), Trichoplusia ni cells ( e.g., High FiveTM cells), and Spodoptera frugiperda cells (e.g., Sf21 or Sf9 cells).
  • the host cell is a mammalian cell.
  • mammalian host cells may include, for example, human embryonic kidney cells (e.g., 293 or 293 cells subcloned for growth in suspension culture), Expi293TM cells, CHO cells, baby hamster kidney cells ( e.g., BHK, ATCC CCL 10), mouse sertoli cells ( e.g., TM4 cells), monkey kidney cells ( e.g., CV1 ATCC CCL 70), African green monkey kidney cells ( e.g., VERO-76, ATCC CRL-1587), human cervical carcinoma cells ( e.g., HELA, ATCC CCL 2), canine kidney cells ( e.g., MDCK, ATCC CCL 34), buffalo rat liver cells ( e.g., BRL 3A, ATCC CRL 1442), human lung cells ( e.g., W138, ATCC CCL 75), human liver cells ( e.g., Hep G2, HB 8065), mouse mammary tumor cells (e.g., MMT
  • the method includes a) culturing a host cell (e.g., any of the host cells described herein) comprising an isolated nucleic acid, vector, and/or vector system (e.g., any of the isolated nucleic acids, vectors, and/or vector systems described herein) under conditions such that the host cell expresses the binding protein; and b) isolating the binding protein from the host cell.
  • a host cell e.g., any of the host cells described herein
  • an isolated nucleic acid, vector, and/or vector system e.g., any of the isolated nucleic acids, vectors, and/or vector systems described herein
  • Methods of isolating proteins from cultured host cells are well known to one of ordinary skill in the art, including, for example, by affinity chromatography (e.g ., two step affinity chromatography comprising protein A affinity chromatography followed by size exclusion chromatography).
  • affinity chromatography e.g ., two step affinity chromatography comprising protein A affinity chromatography followed by size exclusion chromatography.
  • the binding proteins can be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays for the detection and quantitation of one or more target antigens.
  • the binding proteins will bind the one or more target antigens with an affinity that is appropriate for the assay method being employed.
  • binding proteins can be labeled with a detectable moiety.
  • the detectable moiety can be any one that is capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety can be a radioisotope, such as 3 H, 14 C, 32 P, 35 S, 125 I, 99 Tc, 111 In, or 67 Ga; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, ⁇ -galactosidase, or horseradish peroxidase.
  • binding proteins are also useful for in vivo imaging.
  • a binding protein labeled with a detectable moiety can be administered to an animal, e.g., into the bloodstream, and the presence and location of the labeled antibody in the host assayed.
  • the binding protein can be labeled with any moiety that is detectable in an animal, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.
  • kits comprising a binding protein and other reagents useful for detecting target antigen levels in biological samples.
  • reagents can include a detectable label, blocking serum, positive and negative control samples, and detection reagents.
  • the kit comprises a composition comprising any binding protein, polynucleotide, vector, vector system, and/or host cell described herein.
  • the kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing a condition (e.g. , HIV infection) and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the label or package insert indicates that the composition is used for preventing, diagnosing, and/or treating the condition of choice.
  • the article of manufacture or kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water
  • Therapeutic or pharmaceutical compositions comprising binding proteins are within the scope of the disclosure.
  • Such therapeutic or pharmaceutical compositions can comprise a therapeutically effective amount of a binding protein, or binding protein-drug conjugate, in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.
  • Acceptable formulation materials are nontoxic to recipients at the dosages and concentrations employed.
  • the pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition.
  • Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emuls
  • compositions will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the binding protein.
  • the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier for injection can be water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
  • Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute.
  • binding protein compositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution. Further, the binding protein can be formulated as a lyophilizate using appropriate excipients such as sucrose.
  • compositions of the disclosure can be selected for parenteral delivery or subcutaneous.
  • the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally.
  • the preparation of such pharmaceutically acceptable compositions is within the skill of the art.
  • the formulation components are present in concentrations that are acceptable to the site of administration.
  • buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
  • the therapeutic compositions for use can be in the form of a pyrogen-free, parenterally acceptable, aqueous solution comprising the desired binding protein in a pharmaceutically acceptable vehicle.
  • a particularly suitable vehicle for parenteral injection is sterile distilled water in which a binding protein is formulated as a sterile, isotonic solution, properly preserved.
  • Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which can then be delivered via a depot injection.
  • Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation.
  • Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.
  • a pharmaceutical composition can be formulated for inhalation.
  • a binding protein can be formulated as a dry powder for inhalation.
  • Binding protein inhalation solutions can also be formulated with a propellant for aerosol delivery.
  • solutions can be nebulized.
  • binding proteins that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules.
  • a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized.
  • Additional agents can be included to facilitate absorption of the binding protein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.
  • Another pharmaceutical composition can involve an effective quantity of binding proteins in a mixture with non-toxic excipients that are suitable for the manufacture of tablets.
  • Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
  • compositions of the disclosure will be evident to those skilled in the art, including formulations involving binding proteins in sustained- or controlled-delivery formulations.
  • Techniques for formulating a variety of other sustained- or controlled-delivery means such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art.
  • Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules.
  • Sustained release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid.
  • Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art.
  • compositions to be used for in vivo administration typically must be sterile. This can be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method can be conducted either prior to, or following, lyophilization and reconstitution.
  • the composition for parenteral administration can be stored in lyophilized form or in a solution.
  • parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the pharmaceutical composition can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
  • Such formulations can be stored either in a ready-to-use form or in a form ( e.g., lyophilized) requiring reconstitution prior to administration.
  • kits for producing a single-dose administration unit can each contain both a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of this disclosure are kits containing single and multi-chambered pre-filled syringes (e.g ., liquid syringes and lyosyringes).
  • the effective amount of a binding protein pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives.
  • One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the binding protein is being used, the route of administration, and the size (body weight, body surface, or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • Dosing frequency will depend upon the pharmacokinetic parameters of the binding protein in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect.
  • the composition can therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages can be ascertained through use of appropriate dose-response data.
  • the route of administration of the pharmaceutical composition is in accord with known methods, e.g. , orally; through injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release systems; or by implantation devices.
  • the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
  • composition can also be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated.
  • a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated.
  • the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.
  • the pharmaceutical compositions can be used to prevent and/or treat HIV infection.
  • the pharmaceutical compositions can be used as a standalone therapy or in combination with standard anti-retroviral therapy.
  • the present disclosure relates to the bindig protein of the present invention for use in a method of preventing and/or treating HIV infection in a patient.
  • the binding protein for use fo the present invention comprises administering to the patient a therapeutically effective amount of at least one of the binding proteins described herein.
  • the at least one binding protein is administered in combination with an anti-retroviral therapy (e.g., an anti-HIV therepy).
  • the at least one binding protein is administered before the anti-retroviral therapy.
  • the at least one binding protein is administered concurrently with the anti-retroviral therapy.
  • the at least one binding protein is administered after the anti-retroviral therapy.
  • the at least one binding protein is co-administered with any standard anti-retroviral therapy known in the art. In some embodiments, administration of the at least one binding protein results in neutralization of one or more HIV virions. In some embodiments, administration of the at least one binding protein results in elimination of one or more latently and/or chronically HIV-infected cells in the patient. In some embodiments, administration of the at least one binding protein results in neutralization of one or more HIV virions and results in elimination of one or more latently and/or chronically HIV-infected cells in the patient. In some embodiments, the patient is a human.
  • Example 1 production of trispecific binding proteins targeting the HIV-1 Env glycoprotein.
  • the HIV-1 envelope glycoprotein (Env/gp160) is located on the surface of the virus particle, and is composed of a homo-trimer comprising three non-covalently-linked transmembrane gp41 and gp120 complexes.
  • Env enables viral entry into target cells by the binding of gp120 to HIV's main receptor (CD4) and co-receptor (CCR5 or CXCR4), followed by the induction of viral/cellular membrane fusion facilitated by conformational changes in gp41, resulting in entry of the viral capsid and delivery of the viral genome into the host cell. Additionally, Env is expressed on the surface of infected cells.
  • Env acts as the only target for neutralizing antibodies on the HIV-1 virion. Binding of neutralizing antibodies to viral Env inhibits viral attachment/entry. Moreover, binding of neutralizing antibodies to HIV-1 infected, Env expressing cells leads to their destruction by Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Complement Dependent Cytotoxicity (CDC), resulting in reduction of the latent viral reservoir. Thus, neutralizing antibodies targeting Env are an attractive area for anti-viral therapy development. However, because of the high sequence diversity and mutation rate of the HIV-1 virus, developing neutralizing antibodies targeting Env has proven challenging due to the high likelihood that a given HIV-1 strain either lacks the epitope of any given neutralizing antibody, or the strain has evolved a mutation to become resistant to the antibody.
  • ADCC Antibody-Dependent Cell-Mediated Cytotoxicity
  • CDC Complement Dependent Cytotoxicity
  • the vectors expressing the trispecific binding proteins were constructed by inserting the designed heavy and light chain genes into a mammalian expression vector. Corresponding heavy and light chain pairing occurred spontaneously, and heterodimer formation was promoted by Knob-in-Hole mutations engineered in the Fc region.
  • Binding proteins were produced in Expi293 cells by cotransfection of four expression plasmids (Life Technologies, Expi293TM Expression System Kit, Cat. No. A14635). Binding proteins were purified using a two-step purification scheme. First, binding proteins were captured on protein A affinity chromatography resin, washed, and then eluted in glycine at pH 3.0. The eluted proteins were then dialyzed in PBS, concentrated, and filtered. The filtered antibodies were further purified using a Superdex 200 SEC column to obtain monomeric binding proteins.
  • Binding affinities of anti-HIV trispecific binding proteins were measured by surface plasmon resonance (SPR) using a Biacore3000 instrument (GE Healthcare).
  • the assay buffer used was HBS-EP (GE Healthcare).
  • the affinity of the indicated proteins for the MPER binding site on the HIV-1 protein gp41 was measured by surface plasmon resonance (SPR) analysis using a Biacore Instrument as follows: binding proteins were first captured on a CM5 chip coupled with anti-human Fc antibody, followed by flow through of varying concentrations (100 nM - 6.25 nM) of the MPER binding peptide (Acetyl-RRRNEQELLELDKWASLWNWFDITNWLWYIRRR-Ttds-Lys-(Biotin)-NH 2 ) (SEQ ID NO: 284) at 30 ⁇ L per minute, and binding was detected by measurement of association for 240 seconds, and dissociation for 300 seconds on a Biacore 3000 at 25°C.
  • SPR surface plasmon resonance
  • HBS-EP buffer was used for sample dilution, as well as running buffer. Regenration of the chip was done with 3 M MgCl 2 at 30 ⁇ L per minute.
  • BIAevaluation software v.4.1 (GE Healthcare) was used. Data were fit globally using a 1:1 Langmuir model with mass transfer. After software-based curve fitting, the ON and OFF reates at each concentration of MPER binding peptide was calculated and used to obtain a binding affinity for each binding protein.
  • the affinity of the indicated proteins for the CD4BS binding site on the HIV-1 protein gp120 was measured by SPR as follows: recombinant HIV-1 gp120 (Thr27-Arg498) protein (HIV-1 /Clade B/C (CN54), ARCO Biosystems (Cat. # GP4-V15227)) was captured on a CM5 chip, followed by flow through of varying concentrations (100 nM - 6.25 nM) of the binding proteins, and binding was detected by measurement of association for 240 seconds, and dissociation for 300 seconds on a Biacore 3000 at 25°C. HBS-EP buffer was used for sample dilution, as well as running buffer.
  • Conformational stability of the trispecific binding proteins was assessed by determining the melting point T m and aggregation onset temperature (T agg ).
  • T m and aggregation onset temperature (T agg ) were also measured by static light scattering (SLS) using a Unit instrument (Unchained Labs). 9 ⁇ L of each sample was loaded undiluted into a multicuvette array. The samples were then heated from 20°C to 95°C at a heating rate of 0.3°C/minute. The barycentric mean (BCM) of the tryptophan fluorescence spectra was used to measure the protein melting curve, and determine the T m values. The 266 nm static light scattering (SLS) signal was used to measure the aggregation curve and determine the T agg . Data analysis was performed using the Unit analysis software v2.1.
  • a novel strategy was developed for improving neutralizing antibody efficacy against HIV-1, while concomitantly limiting the likelihood of viral breakthrough due to high sequence diversity and/or viral mutation.
  • This strategy involved the generation of trispecific binding proteins comprising four polypeptides that formed three antigen binding sites that specifically bind three different epitopes on the HIV Env glycoprotein ( FIG. 1 ). Each antigen binding site comprised the V H and V L domain from a different HIV-1 neutralizing antibody that targeted a distinct epitope on the Env glycoprotein.
  • the trispecific binding proteins contained a first pair of polypeptides that possessed dual variable domains having a cross-over orientation forming two distinct antigen binding sites (called the CODV Ig format), and a second pair of polypeptides, each with a single variable domain that formed a third antigen binding site ( FIGS. 1A and 1B ).
  • the first pair of polypeptides (that possessed the dual variable domains) comprised a first polypeptide having the structure V L2 -Linker-V L1 -Linker-Immunoglobulin light chain constant domain, and a second polypeptide having the structure V H1 -Linker-V H2 -Linker-Immunoglobulin C H1 , hinge, C H2 , and C H3 heavy chain constant domains, resulting in a pair of polypeptides which had a cross over orienation that formed two distinct antigen binding sites: V H1 -V L1 and V H2 -V L2 ( FIGS. 1C and 1D , see light and heavy chains B).
  • the second pair of polypeptides (that each posessed a single variable domain) comprised a first polypeptide having the structure V H3 -Immunoglobulin C H1 , hinge, C H2 , and C H3 heavy chain constant domains, and a second polypeptide having the structure V L3 -Immunoglobulin light chain constant domain, resulting in a pair of polypetpides that formed a third antigen binding site: V H3 -V L3 ( FIGS. 1C and ID, see light and heavy chains A).
  • the trispecific binding proteins were constructed to include an LS mutation.
  • the trispecific binding proteins were constructed such that within one binding protein, one C H3 domain included a knob mutation and the other C H3 domain included a hole mutation to facilitate heterodimerization of the heavy chains ( FIG. 1 ).
  • Three trispecific binding proteins (Binding Proteins 2, 3, and 24) were generated. These trispecific binding proteins were created by grafting onto a trispecific binding protein framework the V H and V L domains isolated from antibodies targeting three distinct epitopes on the HIV-1 Env glycoprotein: MPER Ab (targeting the MPER epitope on gp41), CD4BS Ab “b” (targeting the CD4 Binding Site on gp120), and V1/V2 directed Ab “a” (targeting the V1/V2 domain on gp120).
  • the three trispecific binding proteins, as well as their parental antibodies, were purified over protein A affinity resin ( FIGS. 2A and 3A ) followed by size exclusion chromatography ( FIGS. 2B and 3B ) to obtain monomeric proteins suitable for further characterization. All three trispecific binding proteins were stable and formed monomers at high frequency.
  • bispecific binding proteins were designed based upon the above described CODV Ig format ( See WO 2012/135345 ), using two different V H and V L domains from the same parental antibodies used to create the trispecific binding proteins.
  • the bispecific binding proteins with these specific variable domains did not purify well as monomers, showing significantly increased aggregate formation when compared to the corresponding trispecific binding proteins ( FIG. 4A and 4B ).
  • the binding affinity of the purified trispecific binding proteins was measured for the HIV-1 Env glycoprotein epitopes on gp41 and gp120.
  • the binding affinity for gp41 was measured for the three trispecific binding proteins, as well as the parental MPER antibody, by Biacore assay using the MPER binding peptide ( FIG. 5 ).
  • the binding affinity for the MPER antibody was calculated to be 18.7 nM.
  • the three trispecific binding proteins all had a higher affinity for the MPER binding peptide than did the parental antibody (Table 3), with Binding Protein 2 having an approximately 3.1 fold higher affinity than the MPER antibody (6.05 nM vs. 18.7 nM, respectively).
  • the binding affinity for the CD4 Binding Site on gp120 was measured for the three trispecific binding proteins, as well as the parental CD4BS antibody, by Biacore assay ( FIG. 6 ).
  • the three trispecific binding proteins all had a similar affinity for the CD4 Binding Site when compared to the parental antibody (Table 4).
  • Table 4 Affinity measurements for the CD4BS binding site Antibody ka (1/Ms) kd (1/s) Rmax (RU) KD (M) Chi2 CD4BS Ab "b" 2.79E+05 2.32E-04 31.4 8.30E-10 1.17 Binding Protein 2 2.31E+05 2.41E-04 34.0 1.04E-09 0.74 Binding Protein 3 7.58E+04 2.75E-04 38.2 3.63E-09 0.19 Binding Protein 24 1.46E+05 2.52E-04 41.6 1.73E-09 0.38
  • the trispecific binding proteins were able to bind both of the tested target epitopes on the HIV-1 Env glycoprotein (Table 5). Moreover, all three trispecific binding proteins bound the target epitopes with affinities approximately equal to or exceeding those of their parental antibodies. Binding affinity of the V1/V2 directed Ab "a”, as well as of the V1/V2 directed Ab "a" binding sites within the three trispecific binding proteins 2, 3, and 24 could not be determined by Biacore analysis because this required a specific gp120 protein antigen which was unavailable. Table 5: Summary of binding capabilities of tested binding proteins Sample Binding on gp120? Binding on gp41? MPER Ab No Yes CD4BS Ab "b” Yes No V1/V2 directed Ab "a” No No Binding Protein 2 Yes Yes Binding Protein 3 Yes Yes Binding Protein 24 Yes Yes Yes
  • Neutralization assays were performed using the TZM-bl assay which measures neutralization as a function of reductions in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene expression after a single round of infection with Env-pseudotyped viruses.
  • the assays were performed as described in Marcella Sarzotti-Kelsoe et al., J. Immunological Methods, 409:131-146 (2014 ). The neutralization results of various antibodies are shown in
  • Assay stocks of Env-pseudotyped viruses were produced in 293T/17 cells by co-transfection with two plasmids: an Env expression plasmid and a plasmid expressing the entire HIV-1 genome except for Env. Co-transfection of these plasmids produced infectious pseudovirus particles which were capable of delivering the Tat gene into target cells, but infections with these pseudovirions could not themselves produce infectious viral progeny.
  • TZM-b1 cells also known as JC53BL-13 cells
  • TZM-b1 cells were engineered to express CD4 and CCR5, and to contain integrated reporter genes for firefly luciferase and E. coli ⁇ -galactosidase under the control of an HIV long-terminal repeat.
  • Reporter gene expression was induced in trans by viral Tat protein (delivered by the pseudotyped viruses) soon after single cycle infection. Luciferase activity was quantified as relative luminescence units (RLU), and was directly proportional to the number of infectious virus particles present in the initial inoculum over a wide range of values. Neutralization was measured as a function of decreased Tat-regulated Firefly luciferase (Luc) reporter gene expression after administration of varying concentrations of the indicated binding proteins. Neutralization titers were identified as the protein dilution at which RLUs were reduced by 80% compared to virus control wells after subtraction of background RLUs. The assay was performed in 96-well plates for high throughput capacity, and well-characterized reference strains were utilized for uniformity across studies.
  • RLU relative luminescence units
  • 21 additional trispecific binding proteins targeting three distinct HIV-1 Env glycoprotein epitopes were generated and purified as described in Example 1. These 21 additional trispecific binding proteins (Binding Proteins 1 and 4-23) were created by grafting onto a trispecific binding protein framework the V H and V L domains isolated from antibodies targeting distinct HIV-1 epitopes on the HIV-1 Env glycoprotein: the anti-MPER antibodies MPER Ab “a” and MPER Ab “b” (targeting the MPER epitope on gp41), the anti-CD4BS antibodies CD4BS Ab “a” and CD4BS Ab “b” (targeting the CD4 Binding Site on gp120), the anti-V1/V2 antibodies V1 / V2 directed Ab "a” and V1 / V2 directed Ab “b” (targeting the V1/V2 domain on gp120), and the anti-V3 antibody V3 directed Ab (targeting the V3 loop on gp120) (Table 7).
  • Binding Protein Epitope Binding Site: 1 MPER x V1/V2 directed / CD4BS 2 MPER x V1/V2 directed / CD4BS 3 V1/V2 directed x MPER / CD4BS 4 MPER x V1/V2 directed / CD4BS 5 MPER x V3 directed / CD4BS 6 V1/V2 directed x MPER / CD4BS 7 V3 directed x V1/V2 directed / CD4BS 8 MPER x V1/V2 directed / CD4BS 9 MPER x V1/V2 directed / CD4BS 10 V1/V2 directed x MPER / CD4BS 11 MPER x V1/V2 directed / CD4BS 12 MPER x V3 directed / CD4BS 13 MPER x V3 directed / V1/V2 directed 14 V1/V2 directed x MPER / CD4BS 15 MPER x V3 directed / V1/V2 directed 16 MPER x V3 directed
  • IC 80 measurements from viral neutralization assav Binding Protein Parental Antibody: 15 1 2 19 20 3 MPER Ab V1/V2 directed Ab "a” V3 directed Ab CD4BS Ab "b” CD4BS Ab "a” # Viruses 208 208 208 208 208 208 208 208 208 208 208 208 208 208 208 208 208 208 208 208 208 208 208 208 208 208 Total VS Neutralized: IC 80 ⁇ 50 ⁇ g/mL 190 202 206 198 206 206 203 151 113 202 183 IC 80 ⁇ 10 ⁇ g/mL 180 199 206 180 206 206 193 149 109 200 175 IC 80 ⁇ 1.0 ⁇ g/mL 166 169 191 145 188 186 61 133 98 184 108 IC 80 ⁇ 0.1 ⁇ g/mL 122 109 136 80 144 123 10 99 72 79 10 IC 80 ⁇
  • Virus Clade KER2008.12.SG3 A 620345.cl.SG3 AE DJ263.8.SG3 AG T266-60.SG3 AG T278-50.SG3 AG BL01.DG.SG3 B BR07.DG.SG3 B CNE57.SG3 B H086.8.SG3 B QH0692.42.SG3 B SS1196.01.SG3 B CNE21.SG3 BC 6471.V1.C16.SG3 C CAP210.E8.SG3 C DU156.12.SG3 C DU422.01.SG3 C TV1.29.SG3 C ZM106.9.SG3 C 3817.v2.c59.SG3 CD X2088.c9.SG3 G Table 10: ICso measurements from viral neutralization assay of 20 representative viruses Total VS Neutralized % VS Neutralized # Viruses IC 80 ⁇ 50 ⁇ g/mL IC 80 ⁇ 1 ⁇ g/mL IC 80 ⁇ 50 ⁇ g/mL IC 80 ⁇ 1
  • PK pharmacokinetics
  • Env is expressed on the surface of HIV-infected cells. Because of this, Env can act as an antibody target to identify infected cells, and induce Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Complement Dependent Cytotoxicity (CDC), resulting in reduction of the latent viral reservoir.
  • ADCC Antibody-Dependent Cell-Mediated Cytotoxicity
  • CDC Complement Dependent Cytotoxicity
  • T cell engagers novel trispecific binding proteins that contain three antigen binding sites targeting three different antigens (HIV-1 Env glycoprotein, CD3, and CD28). These novel proteins not only include antigen binding sites from neutralizing antibodies, but also the ability to bind effector T cells, bringing them into close proximity to infected target cells, thus inducing HIV-infected cell lysis.
  • the binding properties of the T-cell engagers was measured by ELISA assay using a horse radish peroxidase-conjugated anti-Fab probe to detect T-cell engager binding to the surface of ELISA plates coated with CD3, CD28, or Resurfaced Stabilized Core 3 (RSC3) protein of gp120.
  • Human CD3ge-hIgG4 (KIH) (Cat. No: 03-01-0051) from Cambridge Biologics, MA, USA; Human CD28-hIgG4 (Cat. No: 03-01-0303) from Cambridge Biologics, MA, USA.
  • CD4 and CD8 T cell activation were measured as follows: peripheral blood mononuclear cells (PBMCs) were enriched from buffy coats obtained from naive donors (NIH blood bank) using magnetic beads (Miltenyi Biotec). These cells were co-cultured for 14-16 hours with either uninfected or HIV-1 infected CEM cells in the presence of increasing concentrations of the binding proteins (0.01-1.0 ⁇ g/mL) with brefeldin A.
  • PBMCs peripheral blood mononuclear cells
  • NIH blood bank naive donors
  • magnetic beads Magnetic beads
  • the cells were then stained for surface expression of T-cell markers (CD3, CD4, and CD8) and activation markers (CD25 and CD69), followed by intracellular staining for cytokines (IFN- ⁇ , TNF- ⁇ , and IL-2) using fluorescently conjugated antibodies (BD Biosciences, eBiosciences, Biolegend).
  • cytokines IFN- ⁇ , TNF- ⁇ , and IL-2
  • fluorescently conjugated antibodies BD Biosciences, eBiosciences, Biolegend.
  • the number of CD4 and CD8 T cells expressing each cytokine or activation marker was determined by running the samples on an LSRII flow cytometer and analyzing the data with Flowjo software (Treeestar).
  • CD3 downregulation after T cell activation by the T-cell engagers was measured by staining activated PBMCs with non-competing mouse anti-human CD3 antibody and quantitated using flow cytometry.
  • Cytotoxicity of the T-cell engagers to CEM-BaL, ACH2, and J1.1 cells was monitored by flow cytometry as follows: latent cell lines (ACH2, J1.1, OM10) were obtained from the NIH AIDS Reagent Program. The activation of these cells was performed by culturing the cells in the presence or absence of TNF- ⁇ (10 ng/mL) for 14-16 hours. Activation was measured by determining the expression of cell surface HIV envelope glycoprotein by flow cytometry using an allophycocyanin-conjugated anti-HIV Env antibody (2G12).
  • the CEM-IIIb, ACH2, J1.1 and OM10 cells were labeled with the membrane dye PKH-26 (Sigma) and used as target cells in a cytotoxicity assay. These labeled target cells were co-cultured for 14-16 hours at an E:T ratio of 10:1 with enriched human T cells as effector cells in the presence of increasing amounts of the binding proteins. The extent of cell lysis in the target cells was determined by staining with a live/dead cell marker (Life technologies) and measuring the number of dead cells in the labeled target cell population by running the samples on an LSRII flow cytometer followed by analysis using Flowjo software (Treestar).
  • T cell engagers were constructed which contained two antigen binding sites targeting two different T cell surface receptors (CD3 and CD28), and a third antigen binding site targeting the HIV-1 Env glycoprotein ( FIGS. 8A and 8B ).
  • the T cell engagers were constructed to include an LS mutation.
  • the T cell engagers were constructed such that within one binding protein, one C H3 domain included a knob mutation and the other C H3 domain included a hole mutation to facilitate heterodimerization of the heavy chains ( FIGS. 8A and 8B ).
  • T cell engagers Two T cell engagers were constructed which targeted both T cell surface proteins and the HIV-1 Env glycoprotein (Binding Protein 32 and CD3 x CD28 / CD4BS "b" Ab). These two T cell engagers were created by grafting onto a trispecific binding protein framework the V H and V L domains isolated from parental antibodies targeting the T cell surface proteins CD3 and CD28, and the anti-HIV-1 antibody CD4BS Ab "b” (targeting the CD4 Binding Site on gp120).
  • the CD3 x CD28 / CD4BS "b" Ab trispecific binding protein was constructed such that the first pair of polypeptides (which formed two antigen binding sites) targeted the T cell surface receptors CD3 and CD28, and the second pair of polypeptides (which formed the single antigen binding site) targeted the HIV-1 antigen CD4BS ( Table 11 ).
  • Table 11 Format of T-cell engagers Format Name of Construct Arm 1 Antigen Arm 2 Antigen Arm 3 Antigen T cell engagers, trispecific CD3 x CD28 / CD4BS Ab "b" CD4BS CD3 CD28 T cell engagers, trispecific Binding Protein 32 CD4BS CD28 CD3 Monospecific CD4BS IgG4 CD4BS CD4BS -
  • the ability of the two T cell engagers to bind to each of their three target antigens was tested by ELISA assay.
  • the T cell engagers were capable of binding both the CD3 and CD28 T cell surface proteins with the CD3 and CD28 antigen binding sites in either orientation in the bispecific arms of the T cell engagers (i.e., CD3 x CD28 for CD3 x CD28 / CD4BS Ab "b" or CD28 x CD3 for Binding Protein 32). Both T cell engagers were also capable of binding to gp120 (as measured using the HIV-1 RSC3 protein, a gp120 variant lacking the V1, V2, and V3 variable regions) ( FIG. 9 ).
  • T cell activity was next tested for both of the T cell engagers.
  • Incubation of the T cell engagers with monocytes revealed that the T cell engagers induced robust CD8 + T cell activation ( FIG. 10 ).
  • the T cell engagers were capable of inducing significant CD4 + T cell activation on PBMCs alone, or PBMCs incubated with either of the HIV-1 infected T cell lines CEM-NKr cells or CEM-BaL cells ( FIG. 11 ).
  • both of the T cell engagers reduced cell surface expression of CD3 on activated T cells ( FIG. 12 ).
  • the T cell engagers (and positive and negative control bispecific binding proteins targeting CD3 and an HIV antigen) were incubated with the HIV-1 infected T cell line CEM-BaL cells. Incubation of the T cell engagers with the infected cells induced robust cell lysis over a wide range of concentrations ( FIG. 13A ). Likewise, incubation of these T cell engagers induced lysis of the latently infected T cell line ACH2 cells ( FIG. 13B ), as well as J1.1 cells ( FIG. 13C ). Surprisingly, the T cell engagers showed comparable or better cytotoxic activity against chronic and latent HIV-infected cell lines when compared to the bispecific binding proteins.
  • the novel T cell engagers described herein retained the ability from their parental antibodies to bind their target antigens on the HIV-1 Env glycoprotein gp120 on HIV-infected cells, as well as the cell-surface exposed T cell proteins CD3 and CD28.
  • the T cell engagers induced robust CD4 + and CD8 + T cell activation, and diminished CD3 surface expression.
  • these T cell engagers induced significant lysis of HIV-1 infected T cells. Without wishing to be bound by theory, these T cell engagers may provide a novel strategy for anti-viral therapeutics by reducing/eliminating the latent viral reservoir through T cell engagement in HIV/AIDS patients.

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Claims (13)

  1. Protéine de liaison comprenant quatre chaînes polypeptidiques qui forment trois sites de liaison à un antigène qui se lient spécifiquement à au moins une protéine cible du VIH, dans laquelle une première chaîne polypeptidique comprend une structure représentée par la formule :

            VL2- L1-VL1- L2-CL     [I]

    et une deuxième chaîne polypeptidique comprend une structure représentée par la formule :

            VH1- L3-VH2- L4-CH1-hinge-CH2-CH3     [II]

    et une troisième chaîne polypeptidique comprend une structure représentée par la formule :

            VH3-CH1-hinge-CH2-CH3     [III]

    et une quatrième chaîne polypeptidique comprend une structure représentée par la formule :

            VL3-CL     [IV]

    où :
    VL1 est un premier domaine variable de chaîne légère d'immunoglobuline ;
    VL2 est un deuxième domaine variable de chaîne légère d'immunoglobuline ;
    VL3 est un troisième domaine variable de chaîne légère d'immunoglobuline ;
    VH1 est un premier domaine variable de chaîne lourde d'immunoglobuline ;
    VH2 est un deuxième domaine variable de chaîne lourde d'immunoglobuline ;
    VH3 est un troisième domaine variable de chaîne lourde d'immunoglobuline ;
    CL est un domaine constant de chaîne légère d'immunoglobuline ;
    CH1 est un domaine constant de chaîne lourde CH1 d'immunoglobuline ;
    CH2 est un domaine constant de chaîne lourde CH2 d'immunoglobuline ;
    CH3 est un domaine constant de chaîne lourde CH3 d'immunoglobuline ;
    hinge est une région charnière d'immunoglobuline reliant les domaines CH1 et CH2 ; et
    L1, L2, L3 et L4 sont des lieurs d'acides aminés ;
    et dans laquelle le polypeptide de formule I et le polypeptide de formule II forment une paire chaîne légère-chaîne lourde croisée, et
    où :
    (a) VL1 comprend une CDR-L1 comprenant la séquence de SEQ ID N° : 500, une CDR-L2 comprenant la séquence de SEQ ID N° : 501, et une CDR-L3 comprenant la séquence de SEQ ID N° : 274 ; VL2 comprend une CDR-L1 comprenant la séquence de SEQ ID N° : 275, une CDR-L2 comprenant la séquence de SEQ ID N° : 276, et une CDR-L3 comprenant la séquence de SEQ ID N° : 277 ; VL3 comprend une CDR-L1 comprenant la séquence de SEQ ID N° : 266, une CDR-L2 comprenant la séquence de SEQ ID N° : 267, et une CDR-L3 comprenant la séquence de SEQ ID N° : 268 ; VH1 comprend une CDR-H1 comprenant la séquence de SEQ ID N° : 254, une CDR-H2 comprenant la séquence de SEQ ID N° : 255, et une CDR-H3 comprenant la séquence de SEQ ID N° : 256 ; VH2 comprend une CDR-H1 comprenant la séquence de SEQ ID N° : 257, une CDR-H2 comprenant la séquence de SEQ ID N° : 258, et une CDR-H3 comprenant la séquence de SEQ ID N° : 259 ; et VH3 comprend une CDR-H1 comprenant la séquence de SEQ ID N° : 248, une CDR-H2 comprenant la séquence de SEQ ID N° : 497, et une CDR-H3 comprenant la séquence de SEQ ID N° : 250 ; ou
    (b) VL1 comprend une CDR-L1 comprenant la séquence de SEQ ID N° : 500, une CDR-L2 comprenant la séquence de SEQ ID N° : 501, et une CDR-L3 comprenant la séquence de SEQ ID N° : 274 ; VL2 comprend une CDR-L1 comprenant la séquence de SEQ ID N° : 275, une CDR-L2 comprenant la séquence de SEQ ID N° : 276, et une CDR-L3 comprenant la séquence de SEQ ID N° : 277 ; VL3 comprend une CDR-L1 comprenant la séquence de SEQ ID N° : 269, une CDR-L2 comprenant la séquence de SEQ ID N° : 270, et une CDR-L3 comprenant la séquence de SEQ ID N° : 271 ; VH1 comprend une CDR-H1 comprenant la séquence de SEQ ID N° : 254, une CDR-H2 comprenant la séquence de SEQ ID N° : 255, et une CDR-H3 comprenant la séquence de SEQ ID N° : 256 ; VH2 comprend une CDR-H1 comprenant la séquence de SEQ ID N° : 257, une CDR-H2 comprenant la séquence de SEQ ID N° : 258, et une CDR-H3 comprenant la séquence de SEQ ID N° : 259 ; et VH3 comprend une CDR-H1 comprenant la séquence de SEQ ID N° : 251, une CDR-H2 comprenant la séquence de SEQ ID N° : 252, et une CDR-H3 comprenant la séquence de SEQ ID N° : 253 ; ou
    (c) VL1 comprend une CDR-L1 comprenant la séquence de SEQ ID N° : 275, une CDR-L2 comprenant la séquence de SEQ ID N° : 276, et une CDR-L3 comprenant la séquence de SEQ ID N° : 277 ; VL2 comprend une CDR-L1 comprenant la séquence de SEQ ID N° : 500, une CDR-L2 comprenant la séquence de SEQ ID N° : 501, et une CDR-L3 comprenant la séquence de SEQ ID N° : 274 ; VL3 comprend une CDR-L1 comprenant la séquence de SEQ ID N° : 269, une CDR-L2 comprenant la séquence de SEQ ID N° : 270, et une CDR-L3 comprenant la séquence de SEQ ID N° : 271 ; VH1 comprend une CDR-H1 comprenant la séquence de SEQ ID N° : 257, une CDR-H2 comprenant la séquence de SEQ ID N° : 258, et une CDR-H3 comprenant la séquence de SEQ ID N° : 259 ; VH2 comprend une CDR-H1 comprenant la séquence de SEQ ID N° : 254, une CDR-H2 comprenant la séquence de SEQ ID N° : 255, et une CDR-H3 comprenant la séquence de SEQ ID N° : 256 ; et VH3 comprend une CDR-H1 comprenant la séquence de SEQ ID N° : 251, une CDR-H2 comprenant la séquence de SEQ ID N° : 252, et une CDR-H3 comprenant la séquence de SEQ ID N° : 253.
  2. Protéine de liaison selon la revendication 1, dans laquelle :
    (a) VL1 comprend un domaine variable de chaîne légère comprenant la séquence de SEQ ID N° : 518 ; VL2 comprend un domaine variable de chaîne légère comprenant la séquence de SEQ ID N° : 519 ; VL3 comprend un domaine variable de chaîne légère comprenant la séquence de SEQ ID N° : 512 ; VH1 comprend un domaine variable de chaîne lourde comprenant la séquence de SEQ ID N° : 504 ; VH2 comprend un domaine variable de chaîne lourde comprenant la séquence de SEQ ID N° : 506 ; et VH3 comprend un domaine variable de chaîne lourde comprenant la séquence de SEQ ID N° : 502 ; ou
    (b) VL1 comprend un domaine variable de chaîne légère comprenant la séquence de SEQ ID N° : 518 ; VL2 comprend un domaine variable de chaîne légère comprenant la séquence de SEQ ID N° : 519 ; VL3 comprend un domaine variable de chaîne légère comprenant la séquence de SEQ ID N° : 513 ; VH1 comprend un domaine variable de chaîne lourde comprenant la séquence de SEQ ID N° : 504 ; VH2 comprend un domaine variable de chaîne lourde comprenant la séquence de SEQ ID N° : 506 ; et VH3 comprend un domaine variable de chaîne lourde comprenant la séquence de SEQ ID N° : 503 ; ou
    (c) VL1 comprend un domaine variable de chaîne légère comprenant la séquence de SEQ ID N° : 519 ; VL2 comprend un domaine variable de chaîne légère comprenant la séquence de SEQ ID N° : 518 ; VL3 comprend un domaine variable de chaîne légère comprenant la séquence de SEQ ID N° : 513 ; VH1 comprend un domaine variable de chaîne lourde comprenant la séquence de SEQ ID N° : 506 ; VH2 comprend un domaine variable de chaîne lourde comprenant la séquence de SEQ ID N° : 504 ; et VH3 comprend un domaine variable de chaîne lourde comprenant la séquence de SEQ ID N° : 503.
  3. Protéine de liaison selon la revendication 1 ou la revendication 2, dans laquelle :
    (a) le domaine CH3 de la deuxième chaîne polypeptidique comprend des substitutions d'acides aminés à des positions correspondant aux positions 354 et 366 de l'IgG1 humaine selon l'index UE, dans laquelle les substitutions d'acides aminés sont S354C et T366W ; et dans laquelle le domaine CH3 de la troisième chaîne polypeptidique comprend des substitutions d'acides aminés à des positions correspondant aux positions 349, 366, 368, et 407 de l'IgG1 humaine selon l'index UE, dans laquelle les substitutions d'acides aminés sont Y349C, T366S, L368A, et Y407V ; ou
    (b) le domaine CH3 de la deuxième chaîne polypeptidique comprend des substitutions d'acides aminés à des positions correspondant aux positions 349, 366, 368, et 407 de l'IgG1 humaine selon l'index UE, dans laquelle les substitutions d'acides aminés sont Y349C, T366S, L368A, et Y407V ; et dans laquelle le domaine CH3 de la troisième chaîne polypeptidique comprend des substitutions d'acides aminés à des positions correspondant aux positions 354 et 366 de l'IgG1 humaine selon l'index UE, dans laquelle les substitutions d'acides aminés sont S354C et T366W.
  4. Protéine de liaison selon l'une quelconque des revendications 1 à 3, dans laquelle les domaines CH3 des deuxième et troisième chaînes polypeptidiques comprennent des substitutions d'acides aminés à des positions correspondant aux positions 428 et 434 de l'IgG1 humaine selon l'index UE, dans laquelle les substitutions d'acides aminés sont M428L et N434S.
  5. Protéine de liaison selon l'une quelconque des revendications 1 à 4, dans laquelle :
    (a) au moins l'un parmi L1, L2, L3, ou L4 a indépendamment une longueur de 0 acide aminé ; ou
    (b) L1, L2, L3, ou L4 ont chacun indépendamment une longueur d'au moins un acide aminé.
  6. Protéine de liaison selon la revendication 5, dans laquelle L1, L2, L3, et/ou L4 comprennent la séquence Asp- Lys-Thr-His-Thr (SEQ ID N° : 525).
  7. Protéine de liaison comprenant une première chaîne polypeptidique, une deuxième chaîne polypeptidique, une troisième chaîne polypeptidique et une quatrième chaîne polypeptidique dans laquelle :
    (a) la première chaîne polypeptidique comprend la séquence d'acides aminés de SEQ ID N° : 4 ou une séquence d'acides aminés qui est identique à au moins 95 % à la séquence d'acides aminés de SEQ ID N° : 4 ; la deuxième chaîne polypeptidique comprend la séquence d'acides aminés de SEQ ID N° : 3 ou une séquence d'acides aminés qui est identique à au moins 95 % à la séquence d'acides aminés de SEQ ID N° : 3 ; la troisième chaîne polypeptidique comprend la séquence d'acides aminés de SEQ ID N° : 1 ou une séquence d'acides aminés qui est identique à au moins 95 % à la séquence d'acides aminés de SEQ ID N° : 1 ; et la quatrième chaîne polypeptidique comprend la séquence d'acides aminés de SEQ ID N° : 2 ou une séquence d'acides aminés qui est identique à au moins 95 % à la séquence d'acides aminés de SEQ ID N° : 2 ;
    (b) la première chaîne polypeptidique comprend la séquence d'acides aminés de SEQ ID N° : 12 ou une séquence d'acides aminés qui est identique à au moins 95 % à la séquence d'acides aminés de SEQ ID N° : 12 ; la deuxième chaîne polypeptidique comprend la séquence d'acides aminés de SEQ ID N° : 11 ou une séquence d'acides aminés qui est identique à au moins 95 % à la séquence d'acides aminés de SEQ ID N° : 11 ; la troisième chaîne polypeptidique comprend la séquence d'acides aminés de SEQ ID N° : 9 ou une séquence d'acides aminés qui est identique à au moins 95 % à la séquence d'acides aminés de SEQ ID N° : 9 ; et la quatrième chaîne polypeptidique comprend la séquence d'acides aminés de SEQ ID N° : 10 ou une séquence d'acides aminés qui est identique à au moins 95 % à la séquence d'acides aminés de SEQ ID N° : 10 ;
    (c) la première chaîne polypeptidique comprend la séquence d'acides aminés de SEQ ID N° : 20 ou une séquence d'acides aminés qui est identique à au moins 95 % à la séquence d'acides aminés de SEQ ID N° : 20 ; la deuxième chaîne polypeptidique comprend la séquence d'acides aminés de SEQ ID N° : 19 ou une séquence d'acides aminés qui est identique à au moins 95 % à la séquence d'acides aminés de SEQ ID N° : 19 ; la troisième chaîne polypeptidique comprend la séquence d'acides aminés de SEQ ID N° : 17 ou une séquence d'acides aminés qui est identique à au moins 95 % à la séquence d'acides aminés de SEQ ID N° : 17 ; et la quatrième chaîne polypeptidique comprend la séquence d'acides aminés de SEQ ID N° : 18 ou une séquence d'acides aminés qui est identique à au moins 95 % à la séquence d'acides aminés de SEQ ID N° : 18.
  8. Molécule d'acide nucléique isolée comprenant une séquence nucléotidique codant pour la protéine de liaison de l'une quelconque des revendications 1 à 7.
  9. Vecteur d'expression comprenant la molécule d'acide nucléique de la revendication 8.
  10. Système de vecteur comprenant au moins un vecteur codant pour des première, deuxième, troisième, et quatrième chaînes polypeptidiques d'une protéine de liaison de l'une quelconque des revendications 1 à 7, dans lequel éventuellement :
    (a) le système de vecteur comprend un premier vecteur codant pour la première chaîne polypeptidique de la protéine de liaison, un deuxième vecteur codant pour la deuxième chaîne polypeptidique de la protéine de liaison, un troisième vecteur codant pour la troisième chaîne polypeptidique de la protéine de liaison, et un quatrième vecteur codant pour la quatrième chaîne polypeptidique de la protéine de liaison ou
    (b) le système de vecteur comprend un premier vecteur codant pour les première et deuxième chaînes polypeptidiques de la protéine de liaison, et un second vecteur codant pour les troisième et quatrième chaînes polypeptidiques de la protéine de liaison.
  11. Cellule hôte isolée comprenant la molécule d'acide nucléique de la revendication 8, le vecteur d'expression de la revendication 9, ou le système de vecteur de la revendication 10, dans laquelle éventuellement la cellule hôte est une cellule de mammifère ou une cellule d'insecte.
  12. Procédé de production d'une protéine de liaison, le procédé comprenant :
    a) la mise en culture d'une cellule hôte de la revendication 11 dans des conditions telles que la cellule hôte exprime la protéine de liaison ; et
    b) l'isolement de la protéine de liaison de la cellule hôte.
  13. Protéine de liaison de l'une quelconque des revendications 1 à 7 pour une utilisation dans un procédé de prévention et/ou de traitement d'une infection par VIH chez un patient, comprenant l'administration au patient d'une quantité thérapeutiquement efficace de la protéine de liaison, dans laquelle la protéine de liaison est éventuellement administrée conjointement avec une thérapie anti-rétrovirale classique, et dans laquelle le patient est éventuellement un être humain.
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